Evaluation o f the Effec ts o f Cyperm eth rin on Fem ale Rep roduc tive Func tion by Using Rabbit Model and o f the Pro tective Ro le o f Chinese Propo lis

AE Khatab, NM Hashem, LM El-Kodary, FM Lotfy, and GA Hassan

Evaluation o f the Effec ts o f Cyperm eth rin on Fem ale Rep roduc tive Func tion by Using Rabbit Model and o f the Pro tective Ro le o f Chinese Propo lis

AE Khatab1, NM Hashem2,#, LM El-Kodary3, FM Lotfy1, and GA Hassan2

The prophylactic effects of Chinese propolis against cypermethrin toxicity were evaluated by perform ing ovary and uterus histopathology, as well as by characterizing ovarian function, embryos, and litters. Cypermethrin induced atypia in the ovary and uterus, and decreased the ovulation sites and the number of embryos. Cypermethrin-induced oxidative stress during pregnancy, decreased the parturition rate as well as the number and weight of offspring and increased the incidence of morphological malformations in the offspring. Adm inistration of propolis to cypermethrin-treated animalsm itigatedcypermethrin-induced reproductive toxicity.

Cypermethrin is a synthetic form of the naturally derived insecticide pyrethrin, and is used extensively worldwide. It degrades in soil and plants w ithin a few days; however, its concentration stays relatively constant after a treatment indoors, and it can cause hazardous health effects. Cypermethrin can affect reproduction-related steroids, and crosses the placental barrier to interfere with fetal development[1]. Recent studies have shown that the reproductive toxicity of cypermethrin can be partially mediated by oxidative stress[2].

Propolis is a m ixture of resinous plant substances that are produced by honeybees. It containsanabundanceofphytochem icals (flavonoids, phenolic acids, and long-chain fatty acids) w ith antioxidant activities[3]. Therefore, this study aimed to identify the effects of cypermethrin on the different reproductive functions of female rabbits and their offspring, and the protective role of propolis based on its chem ical constituents.

The chem ical constituents of the ethanolic extract of propolis (Guang Zhou Herb & Bee Products Co., Ltd., Tianhe District, Guangzho, China) were identifiedbygaschromatography-mass spectrometry(GC/MS).AThermoScientific TRACE-1300 series GC system fitted with a fused silica DB-5 capillary column (inner diameter, 30 m × 0.32 mm; film thickness, 0.25 μm), coupled to a Triple Quadrupole Mass (TSQ 8000 Evo) was used (Thermo Fisher Scientific Inc., Austin, Texas, USA). The column temperature was set at 40 °C with an initial hold of 5 m in, which was then increased to 270 °C at 2 °C/m in, and maintained at 270 °C for 20 m in. The splitless injection mode was used (0.5 μL of a 1:1000 methanol solution). The carrier gas was helium w ith a flow rate of 1.0 m L/m in. Injector and detector temperatures were 250 °C and 290 °C, respectively. Mass spectra were scanned in the range of 40-700 amu, and the scan time was 5 scans/s. The constituents were identified based on a combination of retention index data and mass spectral data using the Wiley 9 library.

Forty female V-line rabbits (5 months old, weighing 2.935±0.029 kg), obtained from the Laboratory of Rabbit Physiology Research, Faculty of Agriculture, Alexandria University, Egypt, were used. The rabbits were handled in accordance with the Standard Guide for the Care and Use of Laboratory meeting the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes. Propolis (50 mg/kg body weight) and alpha-cypermethrin (50 mg/kg body weight; 1/5 lethal dose, LD20) were dissolved in corn oil. Alphacypermethrin [α-cyano-(3-phenoxyphenyl)methyl(±) -cis/trans-3-(2,2-dichlorovinyl)2,2-dimethylcycloprop -anecarboxylate] is a racem ic m ixture of two isomers of cypermethrin with the molecular formula C22H19Cl2NO3and molecular weight of 416.3 g/mol (Chimac Agriphar S.A., Belgium). Female rabbits were random ly divided into four groups (n=10), and adm inisteredcorn oil (Con group), propolis (Progroup), cypermethrin (Cyp group), or their combination (Cyp/Pro group) by oral gavage. Each female rabbit was treated w ith 25 IU of equine chorionic gonadotropin (Gonaser?, Hipra, Spain) 2 d before being allowed to naturally mate with fertile male rabbits. Female rabbits of each group were divided into two subgroups (5 females each). The first subgroup received the treatments for 60 d, and allowed to mate 2 d prior to being euthanized. Reproductive organs were directly removed and weighed after the animals were euthanized. The number of ovulation points on each ovary was recorded. Excised oviducts were flushed w ith phosphate buffer solution containing 20% bovine serum album in (Sigma, USA). Next, the collected embryos from each oviduct were counted and their developmental stage was recorded. Finally, the ovaries and uteri were fixed and processed for preparation of histological sections. The second subgroup received the same treatmentsimmediatelyaftermatinguntil parturition for two gestation periods. The parturition rate, abortion rate, and offspring characterization data were recorded.

Blood samples of the first subgroup were collected on the day of mating to determ ine the plasma estradiol (E2) concentration. In the second subgroup, blood samples were obtained on days 14 and 28 after mating. The activities of glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) (Reactivos GPL, Barcelona, Spain) and concentrations of malondialdehyde acetate (MDA; Biodiagnostic, Giza, Egypt) and progesterone (P4) were determ ined. Hormonal assessment was carried out using commercial solid-phase enzyme immunoassay kits (DRG International Inc., Springfield, USA).

Results are expressed as mean±standard error values. Analyses of variance (ANOVA) were conducted to determ ine significant differences among groups, followed by Duncan's new multiple range test. Data expressed as percentages were analyzed using a Chi-square test. Statistical significant was considered at P＜0.05. All statistical analyses were carried out using the Statistical Analysis System program (SAS Institute, 2001, Version 8. Cary, USA).

The analysis of the propolis ethanolic extract helped identify 17 chem ical compounds including alkaloids such as 3,3-dimethyl-2-phenyl-2-(1-oxo-1, 2,3,4-tetrahydronaphthalen-2-yl) azirane (21.40%), N,N-dimethyl deacetyl colchinine (3.67%), 2,4-bis (4-chlorophenyl)-5,6-dihydrobenzo[h]quinazoline (3.02%); flavones such as lucenin 2 (5.17%), baicalin (3.82%), and quercetin 7,3',4'-trimethoxy ester (2.71%);andorganosiliconssuchas cyclohexasiloxane, dodecamethyl (6.51%) and hexasiloxane,1,1,3,3,5,5,7,7,9,9,11,11-dodecamethyl (5.41%) (Table 1).

Table 1. Retention Times (RTs, m inute) and Percentages of Relative Area (%) of Chem ical Constituents of Propolis Ethanolic Extract Detected by Gas Chromatography-mass Spectrometry (GC/MS)

As shown in Table 2, the normal diameters of ovarian follicles lined by multiple layers of granulose cells were observed in the Con, Pro, and Cyp/Pro groups. Follicular atresia and fewer layers of granulosa cells were observed in the Cyp group. Compared to the other groups, the Cyp group also showed fewer endometrial glands and increased myometrium hypertrophy.

The highest (P＜0.001) relative uterus weight was observed in the Cyp group. Treatment w ith cypermethrin significantly decreased (P＜0.05) the numberofovulationpointsinthe cypermethrin-treated groups, whereas the number of ovulation points was intermediate in the Cyp/Pro group. The lowest (P＜0.001) concentration of E2was in the Cyp group. However, the combination of propolis w ith cypermethrin significantly improved (P＜0.001) the concentration of E2compared to the cypermethrin-treatedgroup.Treatmentw ith cypermethrin significantly decreased (P＜0.05) the number of embryos, whereas the number of embryos was intermediate in the Cyp/Pro group. Neither the numbers/developmental stages of the embryos collected nor the number of unfertilized ova were affected (P＞0.05) by the treatment.

As shown in Table 3, cypermethrin increased the concentration of MDA (P＜0.01) and decreased the activity of antioxidant enzymes (GPX, CAT, and SOD). The lowest concentration of P4was in the Cyp group, followed by the Cyp/Pro group and Con group. Cypermethrin decreased (P＜0.05) the parturition rate, and increased (P＜0.05) the abortion rate compared to those in the Con, Pro, and Cyp/Pro groups. In addition, it decreased (P＜0.05) the number of offspring born and their birth weights, andincreased(P＜0.05)theincidenceof morphological abnormalities in offspring compared w ith those in the Con, Pro, and Cyp/Pro groups. Propolism itigatedthecypermethrin-induced negative effects on antioxidant enzymes activities, reproductiveperformance,andoffspring characterization.

In this study, cypermethrin caused ovarian follicle atresia and granulosa cell apoptosis, which were associated w ith reductions in the number of ovulation points and the plasma E2concentration. The number of ovulation points depends on the follicle reserves in the ovary. Furthermore, E2issynthesized by granulosa cells, which were damaged by cypermethrin. In a previous study, women exposed to pyrethroids showed decreased ovarian follicle reserves[4]. In this study, cypermethrin caused myometrium hypertrophy, which was associated w ith an increase in the relative weight of the uterus. This finding could be related to the hormone m im icking effect of cypermethrin[1].

Table 2. Histopathology and Relative Weights of Ovaries and Uteri, Ovarian Function, and Embryo Characterization of Female Rabbits Adm inistered Propolis (Pro), Cypermethrin (Cyp), or Their Combination (Cyp/Pro) Compared to Those in the Controls (Con)

Our study showed that cypermethrin decreased the number of embryos, but did not cause deleterious effects in their developmental stage. This seems to be related to decreased ovulation sites rather than fertilization failure. Particularly, the number of unfertilized ova was not affected by cypermethrin. In addition, cypermethrin did not reduce the occurrence of pregnancy (evaluated on day 12 of pregnancy). However, cypermethrin decreased the parturition rate and increased the abortion rate (observed on day 25 of pregnancy). Collectively, the main effects of cypermethrin on pregnancy appeared during the second half of the pregnancy period. The fetal losses m ight be due to the direct effect of cypermethrin on fetus viability, as cypermethrin can cross the placental barrier[1], or due to the destructive effect of free radicals on luteal cell membranes, which reduces P4biosynthesis[5].

The fetal malformation that occurred after the adm inistration of cypermethrin could be partially explainedbythegenotoxicpotentialof cypermethrin (teratogenic effect), which could be mediated by the damaging effect of free radicals on the DNA structure, resulting in gene mutations and thus fetal malformation[6].

Whenpropoliswasadm inistered concom itantly with cypermethrin, it attenuated the reproductivetoxicityofcypermethrinand maintained the activity of antioxidant enzymes. This is most likely due to the unique chem ical constitution of propolis, where high percentages of flavones including lucenin 2, baicalin, and quercetin were detected. Baicalin is suggested to be a strong inducer of neural differentiation[7], and may attenuate the neurotoxicity of cypermethrin. Moreover,luteolinhasantioxidant, anti-inflammatory,antim icrobial,anticancer, anti-allergic, and anti-platelet activities[8]. In addition, alkaloids are known to have anti-inflammatory effects by inhibiting the production of inflammatory mediators such as IL-6, IL-8, and TNF-α[9]. Additionally, organosilicon compounds are used for multi-therapeutic purposes such as the protection of hepatocytes against chem ical toxicity[10].

Table 3. Concentration of Plasma Antioxidant Enzymes and Progesterone (P4), Reproductive Performance, and Offspring Characterization of Female Rabbits Adm inistered Propolis (Pro) or Cypermethrin (Cyp) or Their Combination (Cyp/Pro) Compared to Those in the Control (Con) During Pregnancy (mean±stander error)

Cypermethrin reproductive toxicity could be countered using Chinese propolis as a nutraceutical agent ow ing to its various biological therapeutic properties including antioxidant activity.

#Correspondence should be addressed to NM Hashem, Tel:20-012-88719758,Fax:20-35922780, E-mail: hashemnesreen@yahoo.com

Accepted: October 1, 2016

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10.3967/bes2016.102

1. Department of Home Econom ics Department, Faculty of Specific Education, Alexandria University, Alexandria 21545, Egypt; 2. Department of Animal Production, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria 21545, Egypt; 3. Department of Home Econom ics, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria 21545, Egypt

Biographical note of the AE Khatab, female, born in 1983, PhD student, majoring in maternal and child care.

May 26, 2016;